Inactivated Chimeric Vaccines and Related Methods of Use

ABSTRACT

Embodiments of the present invention generally provide an inactivated chimeric virus vaccine and/or immunogenic composition for the treatment or prevention of viral infection. Further, various other embodiments of the present invention generally relate to methods of preventing and treating virus infection in such animals with the inactivated vaccine and/or immunogenic composition. Other embodiments comprise methods of preparing a vaccine or immunogenic composition for the treatment or prevention of viral infection in such animals.

CROSS REFERENCE TO RELATED APPLICATIONS

This applications claims the benefit under 35 U.S.C. § 119 of U.S.Provisional Application 60/693,629, filed on Jun. 24, 2005, which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention is directed to new and improved methods of preventing andtreating flavivirus and other closely related viral infection inanimals.

BACKGROUND OF THE INVENTION

Flaviviruses are small, enveloped, positive-strand RNA viruses that areof concern in many medical and veterinary settings throughout the world.West Nile virus (WN, or WNV), for example, which is a member of theflavivirus family, is the causative agent of WN encephalitis, aninfectious, non-contagious, arthropod-borne viral disease (In Virology,Fields (ed.), Raven-Lippincott, New York, 1996, pp. 961-1034). The virushas been found in Africa, western Asia, the Middle East, theMediterranean region of Europe, and, recently, in the United States.Mosquitoes become infected with the virus after feeding on infected wildbirds, and then transmit the virus through bites to humans, birds, andanimals, such as horses, sheep, cattle, and pigs.

West Nile virus is an emerging infectious disease. West Nile virus wasfirst isolated in Uganda in 1937. Today it is most commonly found inAfrica, West Asia, Europe, and the Middle East. However, it made itsfirst recognized appearance in the United States in 1999. By 2004, thevirus had been found in birds and mosquitoes in every state exceptAlaska and Hawaii.

Other well-known diseases caused by flaviviruses include yellow fever,Japanese encephalitis, Dengue, and Saint Louis encephalitis. Flavivirusinfections are commonly transmitted by ticks and/or mosquitoes.

The primary hosts for West Nile are only mosquitoes and birds. Otheranimal species, such as humans, and animals, such as horses, sheep,cattle, and pigs, and the like are thought only to be incidental hoststhat become infected when an infected female mosquito bites theincidental host.

People who contract West Nile virus usually experience only mildsymptoms including fever, headache, body aches, skin rash, and swollenlymph glands. If West Nile virus enters the brain, however, it can causelife-threatening encephalitis or meningitis. Life-threatening casesprimarily occur in the elderly. Recent studies have shown that West Nilevirus can be transmitted through blood transfusions and organtransplants. Some health experts also believe it is possible for WestNile virus to be transmitted from a mother to her unborn child, andthrough breast milk.

There are many development projects for West Nile virus vaccineapproaches, including live chimeric vaccines (which combine genes frommore than one virus into a single vaccine), naked DNA vaccines, andvaccines containing cocktails of individual West Nile proteins, and thelike. However, there is no approach making use of an inactivatedchimeric vaccine.

Flavivirus proteins are produced by translation of a single, long openreading frame to generate a polyprotein, which undergoes a complexseries of post-translational proteolytic cleavages by a combination ofhost and viral proteases to generate mature viral proteins. The virusstructural proteins are arranged in the polyprotein in the orderC-prM-E, where “C” is capsid, “prM” is a precursor of the viralenvelope-bound M (membrane) protein, and “E” is the envelope protein.These proteins are present in the N-terminal region of the polyprotein,while the non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, andNS5) are located in the C-terminal region of the polypeptide.

In 2003, human clinical trials of a West Nile live, attenuated virusvaccine were begun by Acambis (Cambridge, Mass.). The Acambis live,attenuated vaccine is based on a vaccine already used for preventingyellow fever, a disease caused by a different flavivirus.

One Acambis live, attenuated vaccine contains genes from two differentviruses, yellow fever and West Nile, and is an example of a chimericvirus. This Acambis live, attenuated vaccine comprises a Yellow Fevervirus with a few genes replaced with genes for surface proteins of WestNile virus.

Details of making this live, attenuated chimeric Acambis vaccine areprovided, for example, in U.S. Pat. Nos. 6,962,708 and 6,696,281 andChambers et al., J. Virol. 73:3095-3101, 1999, which are eachincorporated by reference herein in their entirety. Further methods ofuse and diagnostics for the Acambis live, attenuated chimeric vaccineare provided in U.S. Pat. Nos. 6,682,883 and 6,878,372, which are eachincorporated by reference herein in their entirety.

The results of such live, attenuated vaccines have proven successful andtrials continue. However, certain risks may accompany the use of a live,attenuated virus vaccine. These risks are even more pronounced forimmuno-compromised subjects, the elderly subjects, pregnant subjects,and other subjects with a weakened or stressed immune system. Quiteoften, live, attenuated virus vaccines have been demonstrated to beeither under-attenuated (cause disease) or over-attenuated (fail toimmunize). It is also possible for an optimally-attenuated live virusvaccine to revert to a virulent (disease-causing) form through mutation.However, it should be noted that the YF-WN from Acambis has shown noindication of reversion to virulence. There are additional concerns withlive attenuated vaccines. For example, live Dengue viruses are alsosensitive to heat, making it difficult and costly to maintain thevaccine in some tropical and subtropical countries where the vaccine maybe needed most. Accordingly, a vaccine is needed in the art for safelytreating and/or preventing flavivirus infections, such as West Nile, insubjects with these or other similar risks. Particularly for those whoare immune compromised or other subjects most at risk.

However, the state of the art is that an inactivated chimeric virusvaccine is undesirable and would not be effective. U.S. Pat. No.6,432,411 reported that efforts to make killed flavivirus vaccines havemet with limited success. Primarily the studies were limited by theinability to obtain adequate virus yields from cell culture systems.Virus yields from insect cells are generally in the range of 10⁴ to 10⁵pfu/ml, well below the levels necessary to generate a cost-effectivekilled vaccine. Yields from mammalian cells including LLC-MK2 and Verocells were higher, but the peak yields, approximately 10⁸ pfu/ml from aunique Vero cell line, are still lower than necessary to achieve a trulycost-effective vaccine product.

Accordingly, the art teaches away from the use of inactivatedflaviviruses as viable vaccine candidates. Moreover, there is noteaching of an inactivated chimeric vaccine for treating or preventingany flavivirus infection.

SUMMARY OF THE INVENTION

Various embodiments of the present invention comprise a vaccine orimmunogenic composition for the treatment or prevention of flavivirusinfection in an animal.

The invention also provides methods for preventing or treatingflavivirus infections in susceptible animals, which involveadministering to the subjects inactivated chimeric flaviviruses. Theinvention also provides the use of inactivated chimeric flaviviruses inthe preparation of medicaments for use in such methods and vaccinesand/or immunogenic compositions. In one embodiment of the invention, theinactivated chimeric flaviviruses can include, for example, the capsidand non-structural proteins of a first flavivirus, and the prM andenvelope proteins of a second flavivirus.

In one embodiment, the present invention is directed to an inactivatedchimeric flavivirus, comprising a first flavivirus in which thenucleotide sequences encoding the pre-membrane and envelope proteins arereplaced with nucleotide sequences encoding pre-membrane and envelopeproteins of a second flavivirus. The first flavivirus can be yellowfever virus, including yellow fever virus derived from the 17D strain.The chimeric virus can comprise a signal sequence at the amino acidterminus of the pre-membrane protein, and the signal sequence can bethat of yellow fever virus. The second flavivirus can be West Nilevirus.

In another embodiment, the present invention is directed to animmunogenic composition comprising an inactivated chimeric flavivirus,comprising a first flavivirus in which the nucleotide sequences encodingthe pre-membrane and envelope proteins are replaced with nucleotidesequences encoding pre-membrane and envelope proteins of a secondflavivirus.

In another embodiment, the present invention is directed to a vaccinecomprising an inactivated chimeric flavivirus, comprising a firstflavivirus in which the nucleotide sequences encoding the pre-membraneand envelope proteins are replaced with nucleotide sequences encodingpre-membrane and envelope proteins of a second flavivirus. The vaccinecan further comprise i) one or more modified live viruses; ii) one ormore inactive viruses; or iii) one or more bacterial antigens. Thevaccine can further comprise one or more of inactive Easternencephalomyelitis virus, inactive Western encephalomyelitis virus,inactive Venezuelan encephalomyelitis virus, inactive equine herpesvirus type 1, inactive equine herpes virus type 4, inactive equineinfluenza virus strain Kentucky 1993/A2, inactive equine influenza virusstrain Kentucky 2002/A2, inactive equine influenza virus strain NewMarket/2/93/A2 and a tetanus toxoid fraction.

In another embodiment, the present invention is directed to a method ofpreventing or treating a flavivirus infection in an animal, the methodcomprising administering to the animal an inactivated chimericflavivirus (or immunogenic composition or vaccine thereof) comprising afirst flavivirus in which the nucleotide sequences encoding thepre-membrane and envelope proteins are replaced with nucleotidesequences encoding pre-membrane and envelope proteins of a secondflavivirus. The first flavivirus can be yellow fever virus. The yellowfever virus can be derived from the 17D strain. The second flaviviruscan be West Nile virus.

In any embodiments of the present invention, the inactivated chimericvirus can be present in a concentration ranging between 10² and 10⁸plaque-forming units (pfu). Alternatively, the chimeric flavivirus canbe administered at a dose ranging between 10⁶ and 10⁷ pfu.Alternatively, the chimeric flavivirus can be administered at a doseranging between 1-10 relative antigen dose units.

In any embodiments of the present invention, the inactivated chimericvirus can be administered by a subcutaneous, intramuscular, submucosal,mucosal, or intradermal route. In an embodiment of the presentinvention, the inactivated chimeric flavivirus is orally administered.

Other features and advantages of the invention will be apparent from thefollowing description.

DETAILED DESCRIPTION

As used herein, the term “vaccine(s)” means and refers to a product, theadministration of which is intended to elicit an immune response(s) thatcan prevent and/or lessen the severity of one or more infectiousdiseases. Vaccines can include one or more of the following: a liveattenuated or inactivated preparation of bacteria, viruses or parasites,inactivated (killed) whole organisms, living irradiated cells, crudefractions or purified immunogens, including those derived fromrecombinant DNA in a host cell, conjugates formed by covalent linkage ofcomponents, synthetic antigens, polynucleotides (such as plasmid DNAvaccines), living vectored cells expressing specific heterologousimmunogens, or cells pulsed with immunogen.

As used herein, “chimeric virus” refers to a virus having a genomecontaining sequences from two or more different viruses, includingdifferent viral strains. Unless otherwise stated, “chimera” refers to achimeric virus. A non-limiting example of a chimeric virus is the YF/WNchimera, which is a chimeric flavivirus.

As used herein, “chimeric flavivirus” refers to a virus having a genomecontaining sequences from two or more different flaviviruses, includingdifferent flavivirus strains. As described above, a non-limiting exampleof a chimeric flavivirus is the YF/WN chimera.

As used herein, “West Nile chimeric virus”, “West Nile chimera”, “YF/WNvirus” and “YF/WN chimera” refer to a chimeric live, attenuated virus,comprising the 17D vaccine strain of yellow fever virus (YFV) in whichthe nucleotide sequences encoding the pre-membrane (prM) and envelope(E) proteins are replaced by the nucleotide sequences encoding the prMand E proteins of West Nile Virus (WNV), so that the prM and E proteinsof West Nile virus are expressed, and the capsid protein of the chimericvirus is from the yellow fever virus. The skilled artisan will readilyappreciate that chimeric flaviviruses comprising components of yellowfever virus and West Nile virus can be made other than the specificchimeric flavivirus described in this paragraph.

West Nile chimeric virus (or, YF/WN virus) can be inactivated usingtechniques well known to the skilled artisan. For example, West Nilechimeric virus (or, YF/WN virus) can be inactivated with chemicalinactivating agents or other physical means such as heat. Non-limitingexamples of chemical inactivating agents include binary ethylenimine(BEI) or formalin (a 37% solution of formaldehyde).

Live virus can be inactivated using BEI by first mixing binaryethyleneamine (BEA) powder with a sodium hydroxide solution. After BEIis generated in the basic environment, the BEI solution is added to asolution containing live virus to give a final BEI concentration of 0.5mM to 10 mM. This solution can then be incubated from 4-37° C. for 24-96hours. Sodium thiosulphate can then be added after the virus isinactivated to neutralize any remaining BEI.

Live virus can also be inactivated with formalin (37% solution offormaldehyde). Here, formalin is added to a solution containing livevirus to give a final formalin concentration of 0.05-2% v:v(formalin:live viral solution). This solution can then be incubated from4-37° C. for 24-96 hours.

As used herein, the term “antigen” means and refers to a virus, abacteria, parts of a virus or bacteria or a foreign protein that acts tostimulate the immune system in an animal. The immune system can bestimulated to cause the white blood cells to attack and destroy theantigen or to produce a protein molecule, which attaches to the antigenand either kills the antigen or makes it inactive. As used herein, theterm “antibody” means and refers to a protein-containing molecule thatan animal's immune system makes that reacts with an antigen to make itinactive.

As used herein, the term “animal” means and refers to both human andnon-human animals.

As used herein, the term “vaccine strain” means and refers to a viralstrain suitable for use in an immunogenic composition or vaccine. A“vaccine strain” can comprise, but is not necessarily limited to, anon-pathogenic strain or relatively non-pathogenic strain, a killedstrain, and/or an attenuated strain.

As used herein, the term “lyophilize,” and conjugations thereof, meansand refers to freeze drying. As used herein, the term “animal origin”means and refers to originating from animals. Likewise, the term“non-animal origin” means and refers to not originating directly orindirectly from animals.

As used herein, the term “stabilize,” and conjugations thereof, meansand refers to make or hold stable, firm, steadfast and to maintain atabout a given or substantially unfluctuating level, about a given orsubstantially unfluctuating quality and about a given or substantiallyunfluctuating quantity. However, it is understood that some fluctuationin the level, quality, and/or quantity of the stabilized composition maybe encountered. Embodiments of the present invention are intended toencompass stabilizers that allow such fluctuations. Without limitation,stabilizers include dry stabilizers, bulk stabilizers, cryoprotectants,thermo-stabilizers, osmoprotectants, desiccation protectants, and thelike. Such terms are specifically meant to be included within thestabilizers of the present invention.

As used herein, the term “protein” means and refers to a molecular chainof amino acids. A protein is not of a specific length and can, ifrequired, be modified in vivo or in vitro, by, e.g. glycosylation,amidation, carboxylation or phosphorylation. Inter alia, peptides,oligopeptides and polypeptides are included within the definition ofprotein. A protein or peptide can be of biologic and/or syntheticorigin.

As used herein, the term “nucleic acid” means and refers to a molecularchain of ribonucleic acids or deoxyribonucleic acids. A nucleic acid isnot of a specific length, therefore polynucleotides, genes, open readingframes (ORF's), probes, primers, linkers, spacers and adaptors areincluded within the definition. A nucleic acid can be of biologic and/orsynthetic origin. The nucleic acid may be in single stranded or doublestranded form. The single strand may be in sense or anti-senseorientation. Also included within the definition are modified RNAs orDNAs. Modifications in the bases of the nucleic acid may be made, andbases such as inosine may be incorporated. Other modifications mayinvolve, for example, modifications of the backbone.

As used herein, a pharmaceutically acceptable carrier is understood tobe a compound that does not adversely affect the health of the animal ororganism to be vaccinated, at least not to the extent that the adverseeffect is worse than the effects seen when the animal is not vaccinated.Non-limiting examples of pharmaceutically acceptable carriers includesterile water or a sterile physiological salt solution. In a morecomplex form the carrier can be a buffer.

As used herein, the term “carbohydrate” means and refers to mono-, di-,oligo-, and poly-saccharides.

As used herein, the term “feline” means and refers to any animal of orpertaining to the genus Felis, or family Felidae, cat family, such as,but not limited to, a cat, a lion, a tiger, a mountain lion, a puma, ajaguar, a bobcat, an ocelot and the like.

As used herein, the term “canine” means and refers to any animal of orpertaining to the genus Canis, dog family, such as, but not limited to,a dog, wolf, and the like.

As used herein, the term “equine” means and refers to any animal of orpertaining to the genus Equis, or family Equidae, horse family, such as,but not limited to, a horse, mule, donkey, zebra, and the like.

The present invention generally relates to compositions for and methodsof preventing and treating flavivirus infection in animals. The methodsof the invention involve vaccination of animals that are at risk ofdeveloping or have flavivirus infection with an inactivated chimericflavivirus. Other aspects of the invention are directed to methods ofpreparing a vaccine or immunogenic composition comprising an inactivatedchimeric flavivirus for the treatment or prevention of flavivirusinfection in animals.

The skilled artisan will readily appreciate, however, that there areother well characterized flaviviruses and viruses closely related toflaviviruses that can be treated or prevented using inactivated chimericviruses described by the present invention. Hence, the invention is alsodirected to inactivated chimeric viruses for treating or preventingdiseases or illnesses associated with or caused by viruses of the familyFlaviviridae or Togaviridae. Non-limiting examples of genuses of virusesfalling within these families include viruses belonging to theFlavivirus, Pestivirus, Hepacivirus or Alphavirus genuses. Non-limitingexamples of viruses or diseases caused by viruses belonging to thesefamilies or genuses include encephalitis viruses, Eastern EquineEncephalitis, Western Equine Encephalitis, Venezuela EquineEncephalitis, Kunjin, Murray Valley encephalitis, Louping ill viruses,Japanese encephalitis, Dengue (serotypes 1-4), Yellow Fever, MurrayValley encephalitis, St. Louis encephalitis, Rocio encephalitis,Wesselsbron, Ilheus viruses; tick-borne flaviviruses, such as CentralEuropean encephalitis, Siberian encephalitis, Russian Spring-Summerencephalitis, Kyasanur Forest Disease, Omsk Hemorrhagic fever, Powassan,Negishi, Absettarov, Hansalova, Apoi, and Hypr viruses; as well asviruses from the Hepacivirus genus (e.g., Hepatitis C virus). Additionalviruses that can be treated or prevented using inactivated chimericviruses of the present invention include those belonging to thePestivirus genus (e.g., Bovine diarrhea virus), and other viruses, suchas Lassa, Ebola, and Marburg viruses or other RNA viruses with a genomicconstruction that would be compatible with incorporation into thechimera.

Infection by any of the above described viruses (or diseases causedthereby) can be prevented or treated with the inactivated chimericviruses described herein. In particular, with inactivated chimericviruses that comprise a first virus in which one or more structuralprotein (or proteins) of the first virus has been replaced with acorresponding structural protein (or proteins) of a second virus againstwhich protection or treatment is sought.

A preferred aspect of the invention is directed to inactivated chimericflaviviruses, methods of making inactivated chimeric flaviviruses,vaccines comprising inactivated chimeric flaviviruses, and methods ofusing such vaccines. This aspect of the invention is directed toinactivated chimeric flaviviruses that comprise a flavivirus in whichone or more structural proteins of a first flavivirus have been replacedwith one or more corresponding structural proteins of a secondflavivirus, to which immunity is sought. In one embodiment of thepresent invention, the chimeras consist of the backbone of a firstflavivirus in which the prM and E proteins have been replaced with theprM and E proteins of a second flavivirus.

The inactivated chimeric viruses that are used in the invention canconsist of any combination of viruses, provided that, as is mentionedabove, the virus to which immunity is desired is the source of theinserted structural protein(s). For example, to vaccinate an animal,such as a horse, against West Nile virus infection, a chimericflavivirus consisting of a flavivirus backbone, such as that of yellowfever (YF) virus, into which West Nile virus structural proteins, suchas prM and E proteins, are inserted can be used. In this chimera, the YFprM and E proteins are replaced with those of WN. Similarly, if immunityagainst Japanese encephalitis (JE) virus is desired, then the prM and Eproteins of JE virus can be inserted into a backbone flavivirus, such asa yellow fever virus, in place of the corresponding backbone proteins.Other flaviviruses that cause disease in horses, and for which chimericviruses can be used for inducing protection, include Kunjin, MurrayValley encephalitis, and Louping ill viruses. In all embodiments of thepresent invention, the chimeric virus is then inactivated. Examples ofanimals that can be vaccinated and/or treated with the inactivatedchimeric viruses of the present invention comprise humans, horses, pigs,sheep, cattle, domestic animals, such as cats and dogs, and domesticbirds. However, in general any animal susceptible to infection from theflavivirus for which protection is sought may be vaccinated.

Thus, non-limiting examples of flaviviruses that can be used in theinvention, as sources of backbone virus or structural protein inserts,include mosquito-borne flaviviruses, such as Japanese encephalitis,Dengue (serotypes 1-4), Yellow Fever, Murray Valley encephalitis, St.Louis encephalitis, West Nile, Kunjin, Rocio encephalitis, Wesselsbron,and Ilheus viruses; tick-borne flaviviruses, such as Central Europeanencephalitis, Siberian encephalitis, Russian Spring-Summer encephalitis,Kyasanur Forest Disease, Omsk Hemorrhagic fever, Louping ill, Powassan,Negishi, Absettarov, Hansalova, Apoi, and Hypr viruses; as well asviruses from the Hepacivirus genus (e.g., Hepatitis C virus). Additionalviruses that can be used as the source of inserted structural proteinsinclude viruses from the Pestivirus genus (e.g., Bovine diarrhea virus),and other viruses, such as Lassa, Ebola, and Marburg viruses.

In general, as is disclosed in U.S. Pat. Nos. 6,962,708 and 6,696,281,in an embodiment for the prevention or treatment of West Nile flavivirusinfection, such methods entail replacing genes encoding two structuralproteins [prM and E] of yellow fever 17D vaccine virus with thecorresponding genes of West Nile virus and inactivating the chimericvirus. The resulting inactivated virion has the envelope of West Nile,containing structures involved in virus-cell attachment and virusinternalization, all antigenic determinants for neutralization, andepitope(s) for cytotoxic T lymphocytes. The nucleocapsid (C) protein,nonstructural proteins, and non-translated termini responsible for virusreplication remain those of the original yellow fever 17D virus.

One preferred embodiment of the present invention is directed to aninactivated chimeric vaccine and/or immunogenic composition for thetreatment or prevention of West Nile infection, in an animal susceptibleto West Nile infection. Details of making chimeric viruses including aWN/YF chimeric virus that can then be inactivated and used in variousembodiments of the invention are provided, for example, in U.S. Pat.Nos. 6,962,708 and 6,696,281 and Chambers et al., J. Virol.73:3095-3101, 1999, which are all hereby incorporated by reference intheir entirety. U.S. Pat. Nos. 6,962,708 and 6,696,281 are limited,however, to live attenuated chimeric viruses, vaccines and relatedmethods of use. There is no teaching or suggestion of using aninactivated chimeric virus, use of an inactivated chimeric virus in avaccine, or use of an inactivated chimeric virus in any related method.In contrast to these patents, all embodiments of the present inventionare directed to inactivated chimeric viruses.

Vaccine and immunogenic compositions according to the variousembodiments of the present invention can be prepared and/or marketed inthe form of a liquid, frozen suspension or in a lyophilized form.Typically, vaccines and/or immunogenic compositions prepared accordingto the present invention contain a pharmaceutically acceptable carrieror diluent customarily used for such compositions. Carriers include, butare not limited to, stabilizers, preservatives and buffers. Suitablestabilizers are, for example SPGA, Tween compositions (such as areavailable from A.G. Scientific, Inc., San Diego, Calif.), carbohydrates(such as sorbitol, mannitol, starch, sucrose, dextran, glutamate orglucose), proteins (such as dried milk serum, albumin or casein) ordegradation products thereof. Non-limiting examples of suitable buffersinclude alkali metal phosphates. Suitable preservatives are thimerosal,merthiolate and gentamicin. Diluents include water, aqueous buffer (suchas buffered saline), alcohols and polyols (such as glycerol).

If desired, the inactivated vaccines according to the invention maycontain an adjuvant. Suitable compounds or compositions for this purposeinclude HAVLOGEN® (an acrylic acid polymer-based adjuvant, IntervetInc., Millsboro, Del.), polyacrylic acids, aluminium hydroxide,-phosphate or -oxide, oil-in-water or water-in-oil emulsion based on,for example a mineral oil, such as BAYOL™ or MARCOL™ (Esso Imperial OilLimited, Canada), or a vegetable oil such as vitamin E acetate, andsaponins. However, components with adjuvant activity are widely knownand, generally, any adjuvant may be utilized that does not adverselyinterfere with the efficacy or safety of the vaccine and/or immunogeniccomposition.

Generally, the vaccine may be administered subcutaneously,intradermally, submucosally, or intramuscularly in an effective amountto prevent infection from the flavivirus of interest and/or treat aninfection from the flavivirus. An effective amount is defined as anamount of immunizing inactivated chimeric material that will induceimmunity in the vaccinated animals, against challenge by a virulentvirus. In various other embodiments, an effective amount will induceimmunity in the vaccinated animals or their progeny, against challengeby a virulent virus. Immunity is defined herein as the induction of asignificant higher level of protection in a population of the animalafter vaccination compared to an unvaccinated group.

Further, in various formulations of the inactivated vaccines and/orimmunogenic compositions of the present invention, suitable excipients,stabilizers and the like may be added.

The inactivated chimeric virus can be formulated as a sterile aqueoussolution containing between 10² and 10¹² infectious units (as determinedprior to inactivation). In one embodiment, the inactivated chimericvirus can be formulated as a sterile aqueous solution containing between10⁷ and 10¹⁰ infectious units (as determined prior to inactivation).Infectious units include plaque-forming units (pfu) or tissue cultureinfectious doses (tcid). Alternatively, the inactivated chimeric viruscan be formulated as a sterile aqueous solution containing between 1-10relative antigen dose units. The formulated inactivated chimeric viruscan be provided in a dose volume of 0.1 to 1.0 ml, to be administeredby, for example, subcutaneous, intramuscular, submucosal or intradermalroutes. Further embodiments may be administered by a mucosal route, suchas an oral route. Selection of an appropriate amount of chimera toadminister can be determined by those of skill in this art, and thisamount can vary due to numerous factors, including without limitationthe size, type, and general health of the animal to which the chimera isto be administered.

For a greater understanding of the invention, reference should be madeto the following examples and claims.

EXAMPLE 1 Experimental Design Animals:

Six (6) yearling horses of mixed breed, both male and female, andseronegative to West Nile virus (WNV) were vaccinated with a combinationvaccine containing the inactivated viral components of PRESTIGE® V+VEEvaccine (available from Intervet Inc, Millsboro, Del.) and inactivatedYellow Fever-West Nile (YF-WN) chimera, all combined with a polyacrylicacid adjuvant. PRESTIGE® V+VEE vaccine contains inactivated EasternEncephalomyelitis virus, inactivated Western Encephalomyelitis virus,inactivated Venezuelan Encephalomyelitis virus, inactivated EquineHerpes virus types 1 and 4 (Rhinopneumonitis), inactivated Influenzavirus (Kentucky strain 1993, Kentucky strain 2002, and New Market-2-93),and tetanus toxoid fractions.

The YF-WN live attenuated chimera was obtained from Acambis inCambridge, Mass., and inactivated with binary ethyleneimine (BEI).Inactivation was accomplished by first mixing binary ethyleneamine (BEA)powder with a sodium hydroxide solution. Upon mixing, the BEA convertsto BEI. This BEI liquid solution is added to a solution of live chimericvirus to give a final BEI concentration of 2 mM. The BEI/chimerasolution was incubated at about 18-25° C. for about 3 days.

Another six (6) yearling horses of mixed breed, both male and female,and seronegative to West Nile virus (WNV) were vaccinated with acombination vaccine containing the inactivated viral components ofPRESTIGE® V+VEE vaccine (available from Intervet Inc, Millsboro, Del.)and formalin-inactivated Yellow Fever-West Nile (YF-WN) chimera.

The YF-WN live attenuated chimera was obtained from Acambis inCambridge, Mass., and inactivated with formalin (37% solution offormaldehyde). Inactivation was accomplished by mixing formalin solutionto a solution of live chimeric virus to give a final formalinconcentration of 0.1% v:v with respect to the solution of live chimericvirus. The formalin/chimera solution was incubated at about 18-25° C.for about 3 days.

Another six (6) yearling horses of mixed breed, both male and female,and seronegative to West Nile virus (WNV) were not vaccinated and usedas controls.

Vaccination:

Horses received 2×1 mL dose of vaccine, intramuscular, administered 3-4weeks apart.

Challenge Virus:

West Nile Virus (WNV), 5 log₁₀PFU/1 ml dose, administered per horse bythe intrathecal route at 4 weeks post second vaccination.

Results:

No. Results post challenge with WNV: horses/ ≧102.5° Clin. Vaccine groupF. Signs Viremia Histo. BEI inac. YF/WN virus 6 0/6 0/6 0/6 0/6 Formalininac. YF/WN 6 0/6 0/6 0/6 1/6 virus Unvaccinated controls 6 4/6 5/6 5/65/6

The serological results from Example #1 follow:

Virus neutralizing (VN) antibody titers to WNV in horses vaccinated withtwo dose of inactivated YF-WN or one dose of live YF-WN chimera (fromAcambis, Cambridge, Mass.)

VN titers (50% plaque reduction) on days post vaccination andchallenge^(a) Post Vaccination Post Challenge Vac- Post Post (days)cine^(b) PreVac 1^(st) 2^(nd) 7 14 21 BEI- Negative 80 80 160 >1280 1280inac Negative 40 40 80 640 640 YF-WN Negative 5 20 80 >1280 >2560 virusNegative 5 5 5 1280 1280 Negative 5 80 160 10240 2560 Negative 5 5 3205120 2560 Forma- Negative 160 160 640 >1280 1280 lin Negative 160160 >1280 640 1280 Inac Negative 160 160 320 >1280 1280 YF-WN NegativeNeg 40 320 5120 2560 virus Negative 5 20 20 640 320 Negative 40 40 3202560 2560 Live Negative 160 N/A 320 >1208 1280 YF-WN Negative 160 N/A80 >1280 640 virus Negative 160 N/A 80 >1280 320 Negative 40 N/A 12805120 2560 Negative 5 N/A 80 5120 2560 Negative 640 N/A 1280 20480 5120Control Negative Negative Negative 320 Dead Dead Negative NegativeNegative 80 >1280 >2560 Negative Negative Negative 5 640 640 NegativeNegative Negative 320 2560 1280 Negative Negative Negative 320 Dead DeadNegative Negative Negative 320 Dead Dead ^(a)Titer values are thenumber-fold dilution representing the greatest dilution of serum atwhich 50% plaque reduction is observed relative to control. “Negative”indicates no plaque reduction observed; “80” represents an 80-folddilution; “160” represents a 160-fold dilution, etc.; “N/A” is indicatedwhere no second vaccination was administered; “Dead” refers to thehorses. ^(b)BEI or formalin inactivated YF-WN chimera was in combinationwith PRESTIGE ® V + VEE vaccine.

Results:

The serological results from Example #1 illustrate the unexpectedresults of the inactivated West Nile chimera vaccine of the presentinvention. It is known that live viruses, such as Acambis' liveattenuated YF-WN chimera, produce both cell mediated responses andhumoral responses (antibody response). However, it is generally regardedthat inactivated viruses only produce humoral responses. Here, theinactivated YF-WN produces a high humoral response, as is evident fromthe serological data. Accordingly, and unexpectedly, the inactivatedYF-WN performs as well as the live YF-WN at eliciting a humoralresponse. In addition to the above described advantages of using aninactivated vaccine rather than a live vaccine, the advantages of theinactivated vaccines of the present invention are unexpected.

EXAMPLE 2 Experimental Design Animals: Horses

Six (6) yearling horses of mixed breed, both male and female, andseronegative to West Nile virus (WNV) were vaccinated with a combinationvaccine containing the inactivated viral components of PRESTIGE® V+VEEvaccine (available from Intervet Inc, Millsboro, Del.) and inactivatedYellow Fever-West Nile (YF-WN) chimera. The vaccine also contained apolyacrylic acid adjuvant. PRESTIGE® V+VEE vaccine contains inactivatedEastern Encephalomyelitis virus, inactivated Western Encephalomyelitisvirus, inactivated Venezuelan Encephalomyelitis virus, inactivatedEquine Herpes virus types 1 and 4 (Rhinopneumonitis), inactivatedInfluenza virus (Kentucky strain 1993, Kentucky strain 2002, and NewMarket-2-93), and tetanus toxoid fractions.

The YF-WN live attenuated chimera was obtained from Acambis inCambridge, Mass. The chimeric virus was inactivated with BEI and addedto the PRESTIGE® V+VEE vaccine as described above in Example 1.

Another six (6) yearling horses of mixed breed, both male and female,and seronegative to West Nile virus (WNV) were not vaccinated and usedas controls.

Vaccination

Horses received 2×1 mL dose of vaccine, intramuscular, administered 3-4weeks apart.

Challenge Virus

Horses were challenge by placing 8-17 mosquitoes infected with WNV oneach horse and allowing the mosquitoes to feed for 10-15 minutes.

Results:

No. Results post challenge with WNV: horses/ ≧102.5° Clin. Vaccine groupF. Signs Viremia Histo. Inac. YF/WN 6 0/6 0/6 0/6 0/6 Unvaccinatedcontrols 6 0/6 0/6 5/6 1/6

EXAMPLE 3 I. Experimental Overview

The purpose of this experiment was to establish the immunogenicity ofthe inactivated West Nile chimeric virus contained in a combinationvaccine comprising the antigenic components of PRESTIGE® V+VEE vaccine(available from Intervet Inc, Millsboro, Del.; i.e., inactivated EasternEncephalomyelitis virus, inactivated Western Encephalomyelitis virus,inactivated Venezuelan Encephalomyelitis virus, inactivated EquineHerpes virus types 1 and 4 (Rhinopneumonitis), inactivated Influenzavirus (Kentucky strain 1993, Kentucky strain 2002, and New Market-2-93strain), and Tetanus toxoid), all combined with a polyacrylic acidadjuvant.

In particular, one purpose of this experiment was to establish thenoninterference of the other vaccine fractions with the inactivated WestNile chimeric virus.

Twenty (20) male and female horses were vaccinated two times by theintramuscular (IM) route three to four weeks apart with a 1.0 ml dose ofa combination vaccine comprising inactivated West Nile chimeric virus,inactivated Eastern Encephalomyelitis virus, inactivated WesternEncephalomyelitis virus, inactivated Venezuelan Encephalomyelitis virus,inactivated Equine Herpes virus types 1 and 4 (Rhinopneumonitis),inactivated Influenza virus (Kentucky strain 93, Kentucky strain 2002,and New Market-2-93 strain), and Tetanus toxoid, all combined with apolyacrylic acid adjuvant. Ten additional horses served as unvaccinatedcontrols. At 21 days post-2^(nd) vaccination, vaccinated andunvaccinated control horses were challenged by the intrathecal (IT)route with virulent WNV. Two separate groups of 10 vaccinate and 5control horses were sequentially vaccinated and challenged. Bloodsamples for serological evaluation were collected before vaccination,after vaccination and after challenge and tested for virusneutralization (VN) antibody titers to WNV. Blood samples were collectedpost-challenge for isolation of WNV. Neural tissues were collected atthe time of necropsy for histological examination.

Challenge of horses with virulent WNV by the IT route resulted in signsof neurological disease that are consistent with those observed inhorses infected under natural field conditions. Post-challenge,vaccinated horses showed a statistically significant reduction inclinical signs of neurological disease caused by WNV compared tounvaccinated controls and a statistically significant reduction in virusshedding between vaccinates and controls. These results established thenoninterference of the other vaccine fractions on the Killed FlavivirusChimera fraction. Additional data established the noninterference of theKilled Flavivirus Chimera fraction on the other vaccine fractions.

II. Materials and Methods A. Animals

Thirty (30) horses of mixed sex and breed and six to nine months of agewere used. Horses were identified by a freeze brand. Only horses withvirus neutralizing (VN) antibody titers of ≦5 to WNV as determined by a50% plaque reduction neutralization test were used. Vaccinate andcontrol horses were housed together in insect and rodent prooffacilities during the vaccination period and moved to another facilityfor challenge with virulent WNV.

B. Vaccines

The vaccine contained YF/WN chimera in combination with Easternencephalomyclitis (EE) virus, Western encephalomyelitis (WE) virus,Venezuelan encephalomyelitis (VE) virus, equine herpes virus type 1(EHV-1), equine herpes virus type 4 (EHV-4), equine influenza virus(EIV) strain Kentucky 1993/A2, EIV strain Kentucky 2002/A2, and EIVstrain New Market/2/93/A2, and tetanus toxoid fractions. The vaccinecontained a polyacrylic acid adjuvant.

Two vaccines were used to demonstrate non-interference. One vaccinecontained a minimum immunizing dose of inactivated YF/WN chimera and astandard release dose of the remaining inactivated viruses or tetanustoxoid fractions. The other vaccine contained a standard release dose ofinactivated YF/WN chimera and a minimum immunizing dose of the remaininginactivated viruses or tetanus toxoid fractions. In each case, thecomponent(s) present at the standard release dose did not interfere withthe component(s) present at the minimum immunizing dose level.

C. Vaccination

Horses were 6 to 9 months of age at the time of the first vaccination.Horses were randomized into groups by use of a random number generatorand acclimatized for a minimum of seven days. Twenty horses werevaccinated IM in the neck with two 1 ml doses of the vaccine at threeweeks apart. Ten horses were used as unvaccinated controls. Two groupsof horses (each containing 10 vaccinates and 5 controls) weresequentially vaccinated.

D. Blinding

Project personnel who observed clinical signs and performed laboratorytesting on clinical samples were unaware to which group the horsesbelonged.

F. Observations and Collection of Samples Post-Vaccination

Rectal body temperatures were taken and injection site reactions wereobserved on days −1 through 10 post-vaccination. Body temperatures of≧102.5° F. are considered to be an elevated temperature. Injection sitereactions were scored according to a scoring method. Any systemicreactions or observations of abnormal health were recorded. Blood forserum was collected on days 0, 7, and 21 day post-first vaccination andat 21 days post-second vaccination. Neutralization antibody titers toWNV in serum samples from horses were determined by the use of a 50%plaque reduction neutralization test.

G. Challenge of Horses with Virulent WNV

At 21 days post-second vaccination, horses were challenged by ITadministration of 1 ml containing virulent WNV strain NY99. Results offive replicate titrations of the challenge material were 5.0, 5.1, 5.1,5.0, and 5.0 for a mean of 5.0 and 5.1, 5.1, 5.0, 5.0, and 5.1 for amean of 5.1 log₁₀ PFU/ml dose, for challenge groups 1 and 2,respectively. Rectal body temperatures were recorded on days −1 through21 post-challenge. Challenge of unvaccinated control horses with WNV bythe IT route resulted in clinical signs of disease that are observed inhorses naturally infected with WNV under field conditions. Horses wereobserved during the 21 day post-challenge period for clinical signs ofneurological disease, in the following categories: Changes in mentation,paresis, fasciculations, and ataxia/recumbency. For each category,clinical signs were scored as 0=none, 1=very mild and could gounnoticed, 2=moderate and 3=severe.

Upon confirmation of severe clinical disease, attempts were made toeuthanize the animal within 24 hours. Horses were euthanized for humanereasons due to persistent signs of West Nile disease, or sudden acutesigns coupled with recumbency and/or the inability to locomote withoutassistance as per Center for Veterinary Biologics Notice No. 04-09,dated Apr. 1, 2004. Any other abnormal health observations wererecorded. Blood samples for serology were taken at the time of challengeand at 7, 14, and 21 days post-challenge. Blood samples for virusisolation were taken on days −1 through 10 post-challenge. Blood forserology, virus isolation, and tissues for histopathology were taken atthe time of necropsy. Histopathological lesions in neural tissues werescored as 0=none, 1=very mild/mild, 2=moderate, and 3=severe.

III. Results A. Animals and Vaccination

Body temperatures of ≧102.5° F. were recorded for one day post-firstvaccination for three vaccinate horses and for two days for one controlhorse. Body temperatures of ≧102.5° F. were not recorded for anyvaccinate or control horse after the second vaccination. All horses werein good general health at the start of the study. Vaccination sitereactions were evaluated according to a scoring method (0 (no reaction)to 5 (systemic reaction)). Post-first vaccination, mild injection sitereactions, of scores of 2 or less, were recorded on one or two days forthree horses. Another vaccinate horse had a mild reaction that persistedthrough 10 days post-first vaccination but did not cause any pain orresult in reluctance to move. Mild injection site reactions, of scoresof 2 or less, were also observed post-second vaccination that persistedfor 1 to 6 days post-vaccination. None of the injection site reactionspost-first or second vaccination were noted as painful. No systemicreactions were observed in any of the horses post-first or secondvaccination.

B. Serology Post-Vaccination and Post-Challenge with WNV

Serological data for vaccinated and control horses is summarized in thefollowing tables.

Vaccinate Group Plaque reduction neutralization antibody titers 50%(PRNT50%) to WNV post-1^(st) and 2^(nd) vaccination and post-challenge.

PRNT50% titers to WNV on days post-vaccination and challenge^(a): HorsePost-Vaccination Post-Challenge No. Day 0 Day 7 Day 21^(a) Day 42^(b)Day 7 Day 14 Day 21 201 Neg. Neg. Neg. Neg. ≧1280 ≧1280 ≧1280 205 Neg.Neg. Neg. 40 640 ≧1280 ≧1280 209 Neg. Neg. Neg. 5 5 NS NS 211 Neg. Neg.Neg. 5 80 ≧1280 ≧1280 212 Neg. Neg. Neg. 10 640 ≧1280 ≧1280 214 Neg.Neg. Neg. 20 640 ≧1280 ≧1280 217 Neg. Neg. Neg. 10 ≧1280 ≧1280 ≧1280 218Neg. Neg. Neg. 20 ≧1280 ≧1280 ≧1280 220 Neg. Neg. Neg. Neg. 20 ≧1280≧1280 223 Neg. Neg. Neg. Neg. 320 ≧1280 ≧1280 227 Neg. Neg. Neg. Neg.640 ≧1280 ≧1280 229 Neg. Neg. Neg. Neg. 20 ≧1280 ≧1280 230 Neg. Neg.Neg. 5 160 ≧1280 ≧1280 232 Neg. Neg. Neg. 20 10 ≧1280 ≧1280 233 Neg.Neg. 20 20 20 ≧1280 ≧1280 234 Neg. Neg. Neg. 10 20 ≧1280 ≧1280 235 Neg.Neg. Neg. 5 80 ≧1280 ≧1280 238 Neg. Neg. Neg. 10 40 ≧1280 ≧1280 248 Neg.Neg. Neg. 160 160 ≧1280 ≧1280 254 Neg. Neg. Neg. Neg. 320 ≧1280 ≧1280^(a)Titer values are the number-fold dilution representing the greatestdilution of serum at which 50% plaque reduction is observed relative tocontrol. ^(b)= Day 21 is day of 2^(nd) vaccination, ^(c)= Day 42 is 21days post-2^(nd) vaccination and day of challenge Neg. = Negative, NS =No sample, euthanizedControl Group Plaque reduction neutralization antibody titers 50%(PRNT50%) to WNV post-1^(st) and 2^(nd) vaccination and post-challenge.

PRNT50% titers to WNV on days post-vaccination and challenge^(a): HorsePost-Vaccination Post-Challenge No. Day 0 Day 7 Day 21^(b) Day 42^(c)Day 7 Day 14 Day 21 202 Neg. Neg. Neg. Neg. ≧1280 NS NS 204 Neg. Neg.Neg. Neg. 640 ≧1280 ≧1280 206 Neg. Neg. Neg. Neg. 40 NS NS 208 Neg. Neg.Neg. Neg. 20 640 ≧1280 216 Neg. Neg. Neg. Neg. 20 ≧1280 ≧1280 219 Neg.Neg. Neg. Neg. ≧1280 NS NS 221 Neg. Neg. Neg. Neg. 320 NS NS 225 Neg.Neg. Neg. Neg. 40 ≧1280 ≧1280 231 Neg. Neg. Neg. Neg. 40 ≧1280 ≧1280 239Neg. Neg. Neg. Neg. ≧1280 ≧1280 ≧1280 ^(a)Titer values are thenumber-fold dilution representing the greatest dilution of serum atwhich 50% plaque reduction is observed relative to control. ^(b)= Day 21is day of 2^(nd) vaccination, ^(c)= Day 42 is 21 days post-2^(nd)vaccination and day of challenge Neg. = Negative, NS = No sample,euthanized

All vaccinate and control horses were seronegative to WNV at the time ofvaccination and at 7 days post-first vaccination. The lack of ananammestic response to WNV in vaccinates post-first vaccinationindicated no previous exposure to WNV. Plaque reduction virusneutralizing antibody titers to WNV were detected in one vaccinatepost-first vaccination and were detected in 14 of 20 vaccinates at 21days post-second vaccination. All unvaccinated control horses remainedseronegative throughout the first and second vaccination period. Theseresults demonstrate that the control horses were not exposed to WNVduring the vaccination period, which establishes the validity of thestudy. High levels of virus neutralizing antibody to WNV were detectedin both vaccinate and control horses post-challenge.

C. Rectal Body Temperatures and Neurological Signs in HorsesPost-Challenge with Virulent WNV

Individual body temperatures of horses post-challenge were observed. Sixof the vaccinate horses exhibited body temperatures of ≧102.5° F. forone or two individual days post-challenge and three of these sixvaccinates exhibited body temperatures of ≧102.5° F. for two or moreconsecutive days. Seven of 10 unvaccinated control horses exhibited bodytemperatures of ≧102.5° F. on any day post-challenge and all seven ofthese controls exhibited body temperatures of ≧102.5° F. for two ormores consecutive days. Post-challenge temperatures were compared by arepeated measures analysis of variance using a model that included theeffects of treatment, days, and the interaction of treatment and days.There was a significant difference (P<0.05) in body temperatures betweenvaccinates and control horses on days 8 through 10 post-challenge. Fourof the controls were euthanized by day 10 post-challenge due to theseverity of clinical signs of disease.

Challenge of unvaccinated control horses with WNV by the intrathecal(IT) route resulted in clinical signs of disease that are consistentwith those observed in horses naturally infected with WNV under fieldconditions. Clinical signs that included changes in mentation, paresis,fasciculations, and ataxia/recumbency were also observed.Post-challenge, 7 of 10 (70%) unvaccinated control horses demonstratedmoderate or severe signs of WNV neurological disease for two or moreconsecutive days or demonstrated a severe overall health condition dueto WNV infection such that euthanasia was warranted for humane reasons.Horses were euthanized for humane reasons due to persistent signs ofWest Nile disease, or sudden acute signs coupled with recumbency and/orthe inability to locomote without assistance.

The case definition of infection with WNV and primary outcome fordemonstration of disease caused by WNV was defined as horses havingmoderate or severe signs of disease for two or more consecutive days inany of the categories of: Changes in mentation, paresis, fasciculations,and ataxia/recumbency or any animal in which euthanasia was required dueto overall severe health condition of the animal as a result of WNVinfection. These criteria had to be satisfied in order for the primaryoutcome to be a failure; otherwise, the horse was considered a success.

Only 5 of 20 (25%) of the vaccinated horses demonstrated moderate orsevere signs of WNV neurological disease for two consecutive dayspost-challenge or were euthanized compared to 7 of 10 (70%) of thecontrols. Analysis for the primary outcome was performed in SAS with theFREQ Procedure. Analysis of the proportion of horses meeting the casedefinition showed there was a significant (P<0.02) difference betweenvaccinates and controls. The odds ratio indicated that vaccinated horseswere 6 times more likely to be protected against neurological signs ofWNV disease. A statistically (P<0.05) higher proportion of controls wereeuthanized due to signs of WNV disease compare to controls.

D. Virus Isolation from Serum Post Challenge with Virulent WNV

Results of WNV isolated from serum of horses post challenge were alsoobserved. WNV was recovered from the serum from 6 of 20 vaccinate andfrom 10 of 10 control horses on days 1 through 4 post-challenge.Significantly (P<0.05) more controls were viremic compared to vaccinatesand controls were significantly (P<0.01) viremic more days compared tovaccinates.

E. Histopathology of Neural Tissues in Vaccinates and ControlsPost-Challenge with Virulent WNV

At the time of necropsy, neural tissue from the pons, medulla, andhypothalamus/thalamus were collected and analyzed for histopathology dueto viral encephalitis. In general, there was reduced histopathology invaccinates compared to controls but there was not a statisticallysignificant difference.

IV. Conclusion

Challenge of horses with virulent WNV by the IT route resulted in signsof neurological disease that are consistent with those observed inhorses infected under natural field conditions and are consistent withneurological disease observed in studies with a monovalent vaccine toWNV. Post-challenge, vaccinated horses showed a statisticallysignificant reduction in clinical signs of neurological disease causedby WNV compared to unvaccinated controls and a statistically significantreduction in virus shedding between vaccinates and controls. Theseresults meet the criteria for satisfactory demonstration of theimmunogenicity of the West Nile Virus, Killed Flavivirus Chimerafraction contained in a combination vaccine comprising the components ofPRESTIGE® V+VEE vaccine (available from Intervet Inc, Millsboro, Del.;i.e., Eastern encephalomyelitis (EE) virus, Western encephalomyelitis(WE) virus, Venezuelan encephalomyelitis (VE) virus, equine herpes virustype 1 (EHV-1), equine herpes virus type 4 (EHV-4), equine influenzavirus (EIV) strain Kentucky 1993/A2, EIV strain Kentucky 2002/A2, andEIV strain New Market/2193/A2, and tetanus toxoid fractions). Theseresults established the noninterference of the other vaccine fractionson the Killed Flavivirus Chimera fraction.

While the invention has been described in connection with specificembodiments and examples thereof it will be understood that it iscapable of further modifications and the appended Claims are intended tocover any variations, uses, or adaptations of the invention following,in general the principles of the invention and including such departuresfrom the present disclosure as come within known or customary practicewithin the art to which the invention pertains and as may be applied tothe essential features hereinbefore set forth whether now existing orafter arising. Further, while embodiments of the invention have beendescribed with specific dimensional characteristics and/or measurementsand/or components, it will be understood that the embodiments arecapable of different dimensional characteristics and/or measurementsand/or components without departing from the principles of the inventionand the appended Claims are intended to cover such differences.Furthermore, all patents, printed publications, and the like mentionedherein are hereby incorporated by reference.

1-6. (canceled)
 7. The vaccine of claim 19, wherein the inactivatedchimeric virus is present in a concentration ranging between 10² and 10⁸plaque-forming units (pfu). 8-9. (canceled)
 10. A method of preventingor treating a flavivirus infection in an animal, the method comprisingadministering to the animal an immunogenically effective amount of avaccine comprising an inactivated chimeric flavivirus comprising a firstflavivirus in which the nucleotide sequences encoding the pre-membraneand envelope proteins are replaced with nucleotide sequences encodingpre-membrane and envelope proteins of a second flavivirus, wherein thefirst flavivirus is yellow fever virus and the second flavivirus is WestNile virus. 11-13. (canceled)
 14. The method of claim 10, wherein thechimeric flavivirus is administered at a dose ranging between 10² and10⁸ plaque-forming units (pfu).
 15. The method of claim 14, wherein thechimeric flavivirus is administered at a dose ranging between 10⁶ and10⁷ pfu.
 16. The method of claim 10, wherein the inactivated chimericflavivirus is administered by a subcutaneous, intramuscular, submucosal,mucosal, or intradermal route.
 17. The method of claim 10, wherein theinactivated chimeric flavivirus is orally administered.
 18. (canceled)19. A vaccine comprising an immunogenically effective amount of aninactivated chimeric flavivirus comprising yellow fever virus in whichthe nucleotide sequence encoding the pre-membrane and envelope proteinsare replaced with nucleotide sequences encoding pre-membrane andenvelope proteins of West Nile virus and at lest one additional antigenselected from the croup consisting of inactive Eastern encephalomyelitisvirus, inactive Western encephalomyelitis virus, inactive Venezuelanencephalomyelitis virus, inactive equine herpes virus type 1, inactiveequine herpes virus type 4, inactive equine influenza virus strainKY93/A2, inactive equine influenza virus strain KY02/A2, inactive equineinfluenza virus strain NM/2193/A2 and a tetanus toxoid fraction.
 20. Themethod of claim 10, wherein the vaccine comprising the inactive chimericflavivirus is administered with at least one antigen selected from thegroup consisting of inactive Eastern encephalomyelitis virus, inactiveWestern encephalomyelitis virus, inactive Venezuelan encephalomyelitisvirus, inactive equine herpes virus type 1, inactive equine herpes virustype 4, inactive equine influenza virus strain KY93/A2, inactive equineinfluenza virus strain KY02/A2, inactive equine influenza virus strainNM/2/93/A2 and a tetanus toxoid fraction.